Protecting rnas from degradation using engineered viral rnas

ABSTRACT

This invention is in the field of molecular biology, gene expression, functional genomics, and bioinformatics and relates to novel RNA and related structures and methods of use thereof that enables modulation of gene expression and preservation of particular transcriptome targets. The invention contemplates various applications of RNA sequences derived from the genomic RNA of flaviviruses (FVs) and the application of such features in combination with heterologous sequences.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. Provisional PatentApplication No. 62/270,986, filed on Dec. 22, 2015, which isincorporated herein by reference.

STATEMENT OF GOVERNMENTAL SUPPORT

This invention was made with government support under grant numberGM108257 awarded by National Institutes of Health. The government hascertain rights in the invention.

FIELD OF THE INVENTION

This invention is in the field of molecular biology, gene expression,functional genomics, and bioinformatics and relates to novel RNA andrelated structures and methods of use thereof that enables modulation ofgene expression and preservation of particular transcriptome targets.The invention contemplates various applications of RNA sequences derivedfrom the genomic RNA of flaviviruses (FVs) and the application of suchfeatures in combination with heterologous sequences.

BACKGROUND OF THE INVENTION

The term transcriptome can be applied to the total set of transcripts ina given organism, or to the specific subset of transcripts present in aparticular cell type. Unlike the genome, which is roughly fixed for agiven cell line (excluding mutations), the transcriptome can vary withexternal environmental conditions and internal processes. RNAtranscripts are particularly susceptible to degradation by exonucleases,and this process is used by cells to control the abundance of specifictranscripts in the cell. Because the transcriptome includes all mRNAtranscripts in the cell, the transcriptome reflects the genes that arebeing actively expressed at any given time and the mRNA that is presentin the cell that has not been degraded by the cellular RNA turnoverprocesses. In certain situations, there may be a desire to prolong theexistence of particular RNA transcripts and to specifically target saidtranscript. What are needed are structures and methods for specificallyaltering the transcriptome. Such a method may be useful in applicationsin molecular biology, medicine, and agriculture for example wheremanipulation of the transcriptome could play a role.

SUMMARY OF THE INVENTION

This invention is described in preferred embodiments in the followingdescription with reference to the Figures, in which like numbersrepresent the same or similar elements. Reference throughout thisspecification to “one embodiment,” “an embodiment,” or similar languagemeans that a particular feature, structure, or characteristic describedin connection with the embodiment is included in at least one embodimentof the present invention. Thus, appearances of the phrases “in oneembodiment,” “in an embodiment,” and similar language throughout thisspecification may, but do not necessarily, all refer to the sameembodiment.

The described features, structures, or characteristics of the inventionmay be combined in any suitable manner in one or more embodiments. Inthe following description, numerous specific details are recited toprovide a thorough understanding of embodiments of the invention. Oneskilled in the relevant art will recognize, however, that the inventionmay be practiced without one or more of the specific details, or withother methods, components, materials, and so forth. In other instances,well-known structures, materials, or operations are not shown ordescribed in detail to avoid obscuring aspects of the invention.

This invention is in the field of molecular biology, gene expression,functional genomics, and bioinformatics and relates to novel RNA andrelated structures and methods of use thereof that enables modulation ofgene expression and preservation of particular transcriptome targets.The invention contemplates various applications of RNA sequences derivedfrom the genomic RNA of flaviviruses (FVs) and the application of suchfeatures in combination with heterologous sequences.

In one embodiment, the invention contemplates various applications ofRNA sequences derived from flaviviruses (FVs). These RNA sequences havebeen found to be resistant to cellular enzyme Xrn1, the dominantcytoplasmic 5′→3′ exonuclease. These discrete Xrn1-halting sequences arereferred to as “Xrn1-resistant RNAs” (xrRNAs). The xrRNAs have beenfound to have distinct structural elements and further have certainconserved sequences to retain these resistant structural elements andenable the resultant resistant behavior.

In one embodiment, the invention contemplates that such sequences of RNAcould be useful in a number of applications including, but not limitedto: A) introduction of an xrRNA sequence “in cis” upstream of desiredheterologous mRNA sequences to improve the half-life of the heterologousmRNA sequences by protecting the RNA from exonuclease degradation; andB) provide Xrn1 nuclease resistance by the “in trans” association of anRNA that provides Xrn1-halting ability to an RNA that does not have thisability, thus protecting a “target” RNA from degradation byhybridization of a “protecting” RNA. In this approach, structuressimilar to xrRNAs could be generated by the hybrid association of twoheterologous RNAs rather than placing an xrRNA sequence on at least oneheterologous RNA sequence.

In one embodiment, the invention contemplates that the “in trans”configuration could be used: (1) to alter specific endogenous RNAdegradation rates within cells as a “transcriptome editing” tool and (2)coupling such RNA combinations with a way to turn protected (butuncapped or decapped) mRNAs into viable translation templates as a“translatome editing” tool. It is believed by adjustment of the RNAsequence, that the “protecting RNA” could be “tuned” to achieve variousprotection levels and translation levels of endogenous RNAs. In oneembodiment, the invention contemplates a synthetic ribonucleic acid(RNA) sequence comprising an exonuclease resistant RNA sequence embeddedupstream (5′) of a heterologous RNA sequence, wherein said exonucleaseresistant RNA sequence comprises an interwoven pseudoknot structure. Inone embodiment, said interwoven pseudoknot structure comprises aconserved three-way junction. In one embodiment, said synthetic RNAsequence further comprises an internal ribosome entry site sequencebetween said exonuclease resistant RNA sequence and said heterologousRNA sequence. In one embodiment, said synthetic RNA sequence furthercomprises at least one chemical modification at either the 5′ -end or 3′-end. In one embodiment, said synthetic RNA sequence further comprisesat least one chemical modification at either the 5′ -end or 3′-end.

In one embodiment, the invention contemplates a synthetic ribonucleicacid (RNA) duplex comprising a first exonuclease resistant RNA sequencehybridized to a second heterologous RNA sequence, wherein a region ofsaid first exonuclease resistant RNA sequence and a region of saidsecond heterologous RNA sequence comprise an interwoven pseudoknotstructure that forms an exonuclease-resistant structure. In oneembodiment, said interwoven pseudoknot structure comprises a conservedthree-way junction. In one embodiment, said second heterologous RNAsequence comprises a naturally occurring RNA sequence. In oneembodiment, said first exonuclease resistant RNA sequence furthercomprises a heterologous RNA sequence ligated or attached to the 3′ endof said exonuclease resistant RNA sequence. In one embodiment, saidheterologous RNA sequence comprises small molecule sensing riboswitch.In one embodiment, said first sequence further comprises a translationinitiation element. In one embodiment, said riboswitch disrupts theinterwoven pseudoknot structure in the presence of said small molecule.In one embodiment, said heterologous RNA sequence comprises an openreading frame. In one embodiment, said heterologous RNA sequencecomprises a protein binding sequence. In one embodiment, saidheterologous RNA sequence comprises a Spinach sequence or other similarRNA sequence capable of fluorescence-based visualization. In oneembodiment, said synthetic RNA duplex comprises a chemical modificationof either the 5′ end or 3′ end. In one embodiment, said synthetic RNAduplex comprises at least one chemically modified nucleotide.

Other objects, advantages, and novel features, and further scope ofapplicability of the present invention will be set forth in part in thedetailed description to follow, taken in conjunction with theaccompanying drawings, and in part will become apparent to those skilledin the art upon examination of the following, or may be learned bypractice of the invention. The objects and advantages of the inventionmay be realized and attained by means of the instrumentalities andcombinations particularly pointed out in the appended claims.

Definitions

To facilitate the understanding of this invention, a number of terms aredefined below. Terms defined herein have meanings as commonly understoodby a person of ordinary skill in the areas relevant to the presentinvention. Terms such as “a”, “an” and “the” are not intended to referto only a singular entity, but include the general class of which aspecific example may be used for illustration. The terminology herein isused to describe specific embodiments of the invention, but their usagedoes not delimit the invention, except as outlined in the claims.

As used herein, the term “transcriptome” is used throughout thespecification to describe the set of all RNA molecules, including mRNA,rRNA, tRNA, and other non-coding RNA transcribed in one cell or apopulation of cells. It differs from the exome in that it includes onlythose RNA molecules found in a specified cell population, and usuallyincludes the amount or concentration of each RNA molecule in addition tothe molecular identities.

As used herein, the term “exonuclease” is used throughout thespecification to describe an enzyme that degrades nucleic acid in adirectional manner; that is by interacting with one end of a nucleicacid molecule and degrading it in a stepwise (one nucleotide at a time)manner by progressing to the other end of the molecule (either 5′→3′ or3′→5′).

As used herein, the term “endonuclease” is used throughout thespecification to describe an enzyme that cleaves a nucleic acidinternally; that is, by separating nucleotides within the sequence andnot at the ends of the nucleic acid chain.

As used herein, the term “internal ribosome entry site” or “IRES” isused throughout the specification to describe a nucleotide sequence thatallows for translation initiation in the middle of a messenger RNA(mRNA) sequence. Thus, IRESs drive cap- and end-independent translationof an mRNA.

As used herein, the term “pseudoknot” is used throughout thespecification to describe an RNA base-pairing structural scheme in whichan RNA loop is base-paired to a region located outside (upstream ordownstream) of the stem that flanks or creates the loop.

The terms “coupled”, “connected”, “attached”, “linked”, or “conjugated”are used interchangeably herein and encompass direct as well as indirectconnection, attachment, linkage, or conjugation unless the contextclearly dictates otherwise. The attachment of a ligand to a bead may becovalent or non-covalent. Attachment may be reversible or irreversible.Such attachment includes, but is not limited to, covalent bonding, ionicbonding, Van der Waals forces or friction, and the like.

The term “a means for detecting” as used herein, refers to any methodand/or device that is capable of individually sensing each subset of asolid particle population, even if the subset comprises a single solidparticle. For example, the means may be a flow cytometer that detectsthe solid particles using laser scanning

The term “disease”, as used herein, refers to any impairment of thenormal state of the living animal or plant body or one of its parts thatinterrupts or modifies the performance of the vital functions. Typicallymanifested by distinguishing signs and symptoms, it is usually aresponse to: i) environmental factors (as malnutrition, industrialhazards, or climate); ii) specific infective agents (as worms, bacteria,or viruses); iii) inherent defects of the organism (as geneticanomalies); and/or iv) combinations of these factors.

The term “affinity” as used herein, refers to any attractive forcebetween substances or particles that causes them to enter into andremain in chemical combination. For example, an inhibitor compound thathas a high affinity for a receptor will provide greater efficacy inpreventing the receptor from interacting with its natural ligands, thanan inhibitor with a low affinity.

The term “derived from” as used herein, refers to the source of acompound or sequence. In one respect, a compound or sequence may bederived from an organism or particular species. In another respect, acompound or sequence may be derived from a larger complex or sequence.

The term “protein” as used herein, refers to any of numerous naturallyoccurring extremely complex substances (as an enzyme or antibody) thatconsist of amino acid residues joined by peptide bonds, contain theelements carbon, hydrogen, nitrogen, oxygen, usually sulfur. In general,a protein comprises amino acids having an order of magnitude within thehundreds.

The term “peptide” as used herein, refers to any of various amides thatare derived from two or more amino acids by combination of the aminogroup of one acid with the carboxyl group of another and are usuallyobtained by partial hydrolysis of proteins. In general, a peptidecomprises amino acids having an order of magnitude with the tens.

The term, “purified” or “isolated”, as used herein, may refer to apeptide composition that has been subjected to treatment (i.e., forexample, fractionation) to remove various other components, and whichcomposition substantially retains its expressed biological activity.Where the term “substantially purified” is used, this designation willrefer to a composition in which the protein or peptide forms the majorcomponent of the composition, such as constituting about 50%, about 60%,about 70%, about 80%, about 90%, about 95% or more of the composition(i.e., for example, weight/weight and/or weight/volume). The term“purified to homogeneity” is used to include compositions that have beenpurified to ‘apparent homogeneity” such that there is single proteinspecies (i.e., for example, based upon SDS-PAGE or HPLC analysis). Apurified composition is not intended to mean that some trace impuritiesmay remain.

As used herein, the term “substantially purified” refers to molecules,either nucleic or amino acid sequences, that are removed from theirnatural environment, isolated or separated, and are at least 60% free,preferably 75% free, and more preferably 90% free from other componentswith which they are naturally associated. An “isolated polynucleotide”is therefore a substantially purified polynucleotide.

“Nucleic acid sequence” and “nucleotide sequence” as used herein referto an oligonucleotide or polynucleotide, and fragments or portionsthereof, and to DNA or RNA of genomic or synthetic origin which may besingle- or double-stranded, and represent the sense or antisense strand.

The term “an isolated nucleic acid”, as used herein, refers to anynucleic acid molecule that has been removed from its natural state(e.g., removed from a cell and is, in a preferred embodiment, free ofother genomic nucleic acid).

The terms “amino acid sequence” and “polypeptide sequence” as usedherein, are interchangeable and to refer to a sequence of amino acids.

As used herein the term “portion” when in reference to a protein (as in“a portion of a given protein”) refers to fragments of that protein. Thefragments may range in size from four amino acid residues to the entireamino acid sequence minus one amino acid.

The term “portion” when used in reference to a nucleotide sequencerefers to fragments of that nucleotide sequence. The fragments may rangein size from five nucleotide residues to the entire nucleotide sequenceminus one nucleic acid residue.

The term “antibody” refers to immunoglobulin evoked in animals by animmunogen (antigen). It is desired that the antibody demonstratesspecificity to epitopes contained in the immunogen. The term “polyclonalantibody” refers to immunoglobulin produced from more than a singleclone of plasma cells; in contrast, “monoclonal antibody” refers toimmunoglobulin produced from a single clone of plasma cells.

The terms “specific binding” or “specifically binding” when used inreference to the interaction of an antibody and a protein or peptidemeans that the interaction is dependent upon the presence of aparticular structure (i.e., for example, an antigenic determinant orepitope) on a protein; in other words an antibody is recognizing andbinding to a specific protein structure rather than to proteins ingeneral. For example, if an antibody is specific for epitope “A”, thepresence of a protein containing epitope A (or free, unlabelled A) in areaction containing labeled “A” and the antibody will reduce the amountof labeled A bound to the antibody.

The term “small organic molecule” as used herein, refers to any moleculeof a size comparable to those organic molecules generally used inpharmaceuticals. The term excludes biological macromolecules (e.g.,proteins, nucleic acids, etc.). Preferred small organic molecules rangein size from approximately 10 Da up to about 5000 Da, more preferably upto 2000 Da, and most preferably up to about 1000 Da.

As used herein, the term “antisense” is used in reference to RNAsequences, which are complementary to a specific RNA sequence (e.g.,mRNA). Antisense RNA may be produced by any method, including synthesisby splicing the gene(s) of interest in a reverse orientation to a viralpromoter, which permits the synthesis of a coding strand. Onceintroduced into a cell, this transcribed strand combines with naturalmRNA produced by the cell to form duplexes. These duplexes then blockeither the further transcription of the mRNA or its translation. In thismanner, mutant phenotypes may be generated. The term “antisense strand”is used in reference to a nucleic acid strand that is complementary tothe “sense” strand. The designation (−) (i.e., “negative”) is sometimesused in reference to the antisense strand, with the designation (+)sometimes used in reference to the sense (i.e., “positive”) strand.

As used herein, the terms “siRNA” refers to either small interferingRNA, short interfering RNA, or silencing RNA. Generally, siRNA comprisesa class of double-stranded RNA molecules, approximately 20-25nucleotides in length. Most notably, siRNA is involved in RNAinterference (RNAi) pathways and/or RNAi-related pathways, wherein thecompounds interfere with gene expression.

As used herein, the term “shRNA” refers to any small hairpin RNA orshort hairpin RNA. Although it is not necessary to understand themechanism of an invention, it is believed that any sequence of RNA thatmakes a tight hairpin turn can be used to silence gene expression viaRNA interference. Typically, shRNA uses a vector stably introduced intoa cell genome and is constitutively expressed by a compatible promoter.The shRNA hairpin structure may also cleaved into siRNA, which may thenbecome bound to the RNA-induced silencing complex (RISC). This complexbinds to and cleaves mRNAs which match the siRNA that is bound to it.

As used herein, the term “microRNA”, “miRNA”, or “_(I)IRNA” refers toany single-stranded RNA molecules of approximately 21-23 nucleotides inlength, which regulate gene expression. miRNAs may be encoded by genesfrom whose DNA they are transcribed but miRNAs are not translated intoprotein (i.e. they are non-coding RNAs). Each primary transcript (apri-miRNA) is processed into a short stem-loop structure called apre-miRNA and finally into a functional miRNA. Mature miRNA moleculesare partially complementary to one or more messenger RNA (mRNA)molecules, and their main function is to down-regulate gene expression.

The term “sample” as used herein is used in its broadest sense andincludes environmental and biological samples. Environmental samplesinclude material from the environment such as soil and water. Biologicalsamples may be animal, including, human, fluid (e.g., blood, plasma andserum), solid (e.g., stool), tissue, liquid foods (e.g., milk), andsolid foods (e.g., vegetables). For example, a pulmonary sample may becollected by bronchoalveolar lavage (BAL) which comprises fluid andcells derived from lung tissues. A biological sample may comprise acell, tissue extract, body fluid, chromosomes or extrachromosomalelements isolated from a cell, genomic DNA (in solution or bound to asolid support such as for Southern blot analysis), RNA (in solution orbound to a solid support such as for Northern blot analysis), cDNA (insolution or bound to a solid support) and the like.

As used herein, the terms “complementary” or “complementarity” are usedin reference to “polynucleotides” and “oligonucleotides” (which areinterchangeable terms that refer to a sequence of nucleotides) relatedby the base-pairing rules. For example, the sequence “C-A-G-T,” iscomplementary to the sequence “G-T-C-A.” Complementarity can be“partial” or “total.”“Partial” complementarity is where one or morenucleic acid bases is not matched according to the base pairing rules.“Total” or “complete” complementarity between nucleic acids is whereeach and every nucleic acid base is matched with another base under thebase pairing rules. The degree of complementarity between nucleic acidstrands has significant effects on the efficiency and strength ofhybridization between nucleic acid strands. This is of particularimportance in amplification reactions, as well as detection methods,which depend upon binding between nucleic acids.

The terms “homology” and “homologous” as used herein in reference tonucleotide sequences refer to a degree of complementarity with othernucleotide sequences. There may be partial homology or complete homology(i.e., identity). A nucleotide sequence which is partiallycomplementary, i.e., “substantially homologous,” to a nucleic acidsequence is one that at least partially inhibits a completelycomplementary sequence from hybridizing to a target nucleic acidsequence. The inhibition of hybridization of the completelycomplementary sequence to the target sequence may be examined using ahybridization assay (Southern or Northern blot, solution hybridizationand the like) under conditions of low stringency. A substantiallyhomologous sequence or probe will compete for and inhibit the binding(i.e., the hybridization) of a completely homologous sequence to atarget sequence under conditions of low stringency. This is not to saythat conditions of low stringency are such that non-specific binding ispermitted; low stringency conditions require that the binding of twosequences to one another be a specific (i.e., selective) interaction.The absence of non-specific binding may be tested by the use of a secondtarget sequence, which lacks even a partial degree of complementarity(e.g., less than about 30% identity); in the absence of non-specificbinding, the probe will not hybridize to the second non-complementarytarget.

The terms “homology” and “homologous” as used herein in reference toamino acid sequences refer to the degree of identity of the primarystructure between two amino acid sequences. Such a degree of identitymay be directed to a portion of each amino acid sequence, or to theentire length of the amino acid sequence. Two or more amino acidsequences that are “substantially homologous” may have at least 50%identity, preferably at least 75% identity, more preferably at least 85%identity, most preferably at least 95%, or 100% identity.

An oligonucleotide sequence that is a “homolog” is defined herein as anoligonucleotide sequence that exhibits greater than or equal to 50%identity to a sequence, when sequences having a length of 100 bp orlarger are compared.

Low stringency conditions comprise conditions equivalent to binding orhybridization at 42° C. in a solution consisting of 5× SSPE (43.8 g/1NaCl, 6.9 g/1 NaH2PO4.H2O and 1.85 g/1 EDTA, pH adjusted to 7.4 withNaOH), 0.1% SDS, 5× Denhardt's reagent (50× Denhardt's contains per 500ml: 5 g Ficoll (Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)) and100μg/ml denatured salmon sperm DNA followed by washing in a solutioncomprising 5× SSPE, 0.1% SDS at 42° C. when a probe of about 500nucleotides in length is employed. Numerous equivalent conditions mayalso be employed to comprise low stringency conditions; factors such asthe length and nature (DNA, RNA, base composition) of the probe andnature of the target (DNA, RNA, base composition, present in solution orimmobilized, etc.) and the concentration of the salts and othercomponents (e.g., the presence or absence of formamide, dextran sulfate,polyethylene glycol), as well as components of the hybridizationsolution may be varied to generate conditions of low stringencyhybridization different from, but equivalent to, the above listedconditions. In addition, conditions which promote hybridization underconditions of high stringency (e.g., increasing the temperature of thehybridization and/or wash steps, the use of formamide in thehybridization solution, etc.) may also be used.

As used herein, the term “hybridization” is used in reference to thepairing of complementary nucleic acids using any process by which astrand of nucleic acid joins with a complementary strand through basepairing to form a hybridization complex. Hybridization and the strengthof hybridization (i.e., the strength of the association between thenucleic acids) is impacted by such factors as the degree ofcomplementarity between the nucleic acids, stringency of the conditionsinvolved, the Tm of the formed hybrid, and the G:C ratio within thenucleic acids.

As used herein the term “hybridization complex” refers to a complexformed between two nucleic acid sequences by virtue of the formation ofhydrogen bounds between complementary G and C bases and betweencomplementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., C0 t or R0 tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized to a solid support (e.g., anylon membrane or a nitrocellulose filter as employed in Southern andNorthern blotting, dot blotting or a glass slide as employed in in situhybridization, including FISH (fluorescent in situ hybridization)).

As used herein, the term “Tm” is used in reference to the “meltingtemperature.” The melting temperature is the temperature at which apopulation of double-stranded nucleic acid molecules becomes halfdissociated into single strands. As indicated by standard references, asimple estimate of the Tm value may be calculated by the equation:Tm=81.5+0.41 (% G+C), when a nucleic acid is in aqueous solution at 1MNaCl. Anderson et al., “Quantitative Filter Hybridization” In: NucleicAcid Hybridization (1985) [1]. Computations that are more sophisticatedtake structural, as well as sequence characteristics, into account forthe calculation of Tm.

As used herein the term “stringency” is used in reference to theconditions of temperature, ionic strength, and the presence of othercompounds such as organic solvents, under which nucleic acidhybridizations are conducted. “Stringency” typically occurs in a rangefrom about Tm to about 20° C. to 25° C. below Tm. A “stringenthybridization” can be used to identify or detect identicalpolynucleotide sequences or to identify or detect similar or relatedpolynucleotide sequences. For example, when fragments are employed inhybridization reactions under stringent conditions the hybridization offragments which contain unique sequences (i.e., regions which are eithernon-homologous to or which contain less than about 50% homology orcomplementarity) are favored. Alternatively, when conditions of “weak”or “low” stringency are used hybridization may occur with nucleic acidsthat are derived from organisms that are genetically diverse (i.e., forexample, the frequency of complementary sequences is usually low betweensuch organisms).

As used herein, the term “amplifiable nucleic acid” is used in referenceto nucleic acids which may be amplified by any amplification method. Itis contemplated that “amplifiable nucleic acid” will usually comprise“sample template.”

As used herein, the term “sample template” refers to nucleic acidoriginating from a sample that is analyzed for the presence of a targetsequence of interest. In contrast, “background template” is used inreference to nucleic acid other than sample template that may or may notbe present in a sample. Background template is most often inadvertent.It may be the result of carryover, or it may be due to the presence ofnucleic acid contaminants sought to be purified away from the sample.For example, nucleic acids from organisms other than those to bedetected may be present as background in a test sample.

“Amplification” is defined as the production of additional copies of anucleic acid sequence and is generally carried out using polymerasechain reaction. Dieffenbach C. W. and G. S. Dveksler (1995) In: PCRPrimer, a Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.[2].

As used herein, the term “polymerase chain reaction” (“PCR”) refers tothe method of K. B. Mullis U.S. Pat. No. 4,683,195 [3] and U.S. Pat. No.4,683,202 [4], herein incorporated by reference, which describe a methodfor increasing the concentration of a segment of a target sequence in amixture of genomic DNA without cloning or purification. The length ofthe amplified segment of the desired target sequence is determined bythe relative positions of two oligonucleotide primers with respect toeach other, and therefore, this length is a controllable parameter. Byvirtue of the repeating aspect of the process, the method is referred toas the “polymerase chain reaction” (hereinafter “PCR”). Because thedesired amplified segments of the target sequence become the predominantsequences (in terms of concentration) in the mixture, they are said tobe “PCR amplified”. With PCR, it is possible to amplify a single copy ofa specific target sequence in genomic DNA to a level detectable byseveral different methodologies (e.g., hybridization with a labeledprobe; incorporation of biotinylated primers followed by avidin-enzymeconjugate detection; incorporation of 32P-labeled deoxynucleotidetriphosphates, such as dCTP or dATP, into the amplified segment). Inaddition to genomic DNA, any oligonucleotide sequence can be amplifiedwith the appropriate set of primer molecules. In particular, theamplified segments created by the PCR process itself are, themselves,efficient templates for subsequent PCR amplifications.

As used herein, the term “primer” refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product which is complementary to a nucleic acid strand isinduced, (i.e., in the presence of nucleotides and an inducing agentsuch as DNA polymerase and at a suitable temperature and pH).

The primer is preferably single stranded for maximum efficiency inamplification, but may alternatively be double stranded. If doublestranded, the primer is first treated to separate its strands beforebeing used to prepare extension products. Preferably, the primer is anoligodeoxy-ribonucleotide. The primer must be sufficiently long to primethe synthesis of extension products in the presence of the inducingagent. The exact lengths of the primers will depend on many factors,including temperature, source of primer and the use of the method.

As used herein, the term “probe” refers; to an oligonucleotide (i.e., asequence of nucleotides), whether occurring naturally as in a purifiedrestriction digest or produced synthetically, recombinantly or by PCRamplification, which is capable of hybridizing to anotheroligonucleotide of interest. A probe may be single-stranded ordouble-stranded. Probes are useful in the detection, identification andisolation of particular gene sequences. It is contemplated that anyprobe used in the present invention will be labeled with any “reportermolecule,” so that is detectable in any detection system, including, butnot limited to enzyme (e.g., ELISA, as well as enzyme-basedhistochemical assays), fluorescent, radioactive, and luminescentsystems. It is not intended that the present invention be limited to anyparticular detection system or label.

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence.

DNA molecules are said to have “5′ ends” and “3′ ends” becausemononucleotides are reacted to make oligonucleotides in a manner suchthat the 5′ phosphate of one mononucleotide pentose ring is attached tothe 3′ oxygen of its neighbor in one direction via a phosphodiesterlinkage. Therefore, an end of an oligonucleotide is referred to as the“5′ end” if its 5′ phosphate is not linked to the 3′ oxygen of amononucleotide pentose ring. An end of an oligonucleotide is referred toas the “3′ end” if its 3′ oxygen is not linked to a 5′ phosphate ofanother mononucleotide pentose ring. As used herein, a nucleic acidsequence, even if internal to a larger oligonucleotide, also may be saidto have 5′ and 3′ ends. In either a linear or circular DNA molecule,discrete elements are referred to as being “upstream” or 5′ of the“downstream” or 3′ elements. This terminology reflects the fact thattranscription proceeds in a 5′ to 3′ fashion along the DNA strand. Thepromoter and enhancer elements which direct transcription of a linkedgene are generally located 5′ or upstream of the coding region. However,enhancer elements can exert their effect even when located 3′ of thepromoter element and the coding region. Transcription termination andpolyadenylation signals are located 3′ or downstream of the codingregion.

As used herein, the term “an oligonucleotide having a nucleotidesequence encoding a gene” means a nucleic acid sequence comprising thecoding region of a gene, i.e. the nucleic acid sequence which encodes agene product. The coding region may be present in a cDNA, genomic DNA orRNA form. When present in a DNA form, the oligonucleotide may besingle-stranded (i.e., the sense strand) or double-stranded. Suitablecontrol elements such as enhancers/promoters, splice junctions,polyadenylation signals, etc. may be placed in close proximity to thecoding region of the gene if needed to permit proper initiation oftranscription and/or correct processing of the primary RNA transcript.Alternatively, the coding region utilized in the expression vectors ofthe present invention may contain endogenous enhancers/promoters, splicejunctions, intervening sequences, polyadenylation signals, etc. or acombination of both endogenous and exogenous control elements.

As used herein, the term “regulatory element” refers to a geneticelement that controls some aspect of the expression of nucleic acidsequences. For example, a promoter is a regulatory element thatfacilitates the initiation of transcription of an operably linked codingregion. Other regulatory elements are splicing signals, polyadenylationsignals, termination signals, etc.

The term “in operable combination” as used herein, refers to any linkageof nucleic acid sequences in such a manner that a nucleic acid moleculecapable of directing the transcription of a given gene and/or thesynthesis of a desired protein molecule is produced. Regulatorysequences may be operably combined to an open reading frame includingbut not limited to initiation signals such as start (i.e., ATG) and stopcodons, promoters which may be constitutive (i.e., continuously active)or inducible, as well as enhancers to increase the efficiency ofexpression, and transcription termination signals.

Transcriptional control signals in eukaryotes comprise “promoter” and“enhancer” elements. Promoters and enhancers consist of short arrays ofDNA sequences that interact specifically with cellular proteins involvedin transcription. Maniatis, T. et al., Science 236:1237 (1987) [5].Promoter and enhancer elements have been isolated from a variety ofeukaryotic sources including genes in plant, yeast, insect and mammaliancells and viruses (analogous control elements, i.e., promoters, are alsofound in prokaryotes). The selection of a particular promoter andenhancer depends on what cell type is to be used to express the proteinof interest. The presence of “splicing signals” on an expression vectoroften results in higher levels of expression of the recombinanttranscript. Splicing signals mediate the removal of introns from theprimary RNA transcript and consist of a splice donor and acceptor site.Sambrook, J. et al., In: Molecular Cloning: A Laboratory Manual, 2nded., Cold Spring Harbor laboratory Press, New York (1989) pp. 16.7-16.8.A commonly used splice donor and acceptor site is the splice junctionfrom the 16S RNA of SV40 [6].

The term “poly A site” or “poly A sequence” as used herein denotes a DNAsequence that directs both the termination and polyadenylation of thenascent RNA transcript. Efficient polyadenylation of the recombinanttranscript is desirable as transcripts lacking a poly A tail areunstable and are rapidly degraded. The poly A signal utilized in anexpression vector may be “heterologous” or “endogenous.” An endogenouspoly A signal is one that is found naturally at the 3′ end of the codingregion of a given gene in the genome. A heterologous poly A signal isone which is isolated from one gene and placed 3′ of another gene.Efficient expression of recombinant DNA sequences in eukaryotic cellsinvolves expression of signals directing the efficient termination andpolyadenylation of the resulting transcript. Transcription terminationsignals are generally found downstream of the polyadenylation signal andare a few hundred nucleotides in length.

As used herein, the terms “nucleic acid molecule encoding”, “DNAsequence encoding,” and “DNA encoding” refer to the order or sequence ofdeoxyribonucleotides along a strand of deoxyribonucleic acid. The orderof these deoxyribonucleotides determines the order of amino acids alongthe polypeptide (protein) chain. The DNA sequence thus codes for theamino acid sequence.

The term “Southern blot” refers to the analysis of DNA on agarose oracrylamide gels to fractionate the DNA according to size, followed bytransfer and immobilization of the DNA from the gel to a solid support,such as nitrocellulose or a nylon membrane. The immobilized DNA is thenprobed with a labeled oligodeoxyribonucleotide probe or DNA probe todetect DNA species complementary to the probe used. The DNA may becleaved with restriction enzymes prior to electrophoresis. Followingelectrophoresis, the DNA may be partially depurinated and denaturedprior to or during transfer to the solid support. Southern blots are astandard tool of molecular biologists. J. Sambrook et al. (1989) In:Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y.,pp 9.31-9.58 [6].

The term “Northern blot” as used herein refers to the analysis of RNA byelectrophoresis of RNA on agarose gels to fractionate the RNA accordingto size followed by transfer of the RNA from the gel to a solid support,such as nitrocellulose or a nylon membrane. The immobilized RNA is thenprobed with a labeled oligodeoxyribonucleotide probe or DNA probe todetect RNA species complementary to the probe used. Northern blots are astandard tool of molecular biologists. J. Sambrook, J. et al. (1989)supra, pp 7.39-7.52 [6].

The term “reverse Northern blot” as used herein refers to the analysisof DNA by electrophoresis of DNA on agarose gels to fractionate the DNAon the basis of size followed by transfer of the fractionated DNA fromthe gel to a solid support, such as nitrocellulose or a nylon membrane.The immobilized DNA is then probed with a labeled oligoribonuclotideprobe or RNA probe to detect DNA species complementary to the ribo probeused.

As used herein the term “coding region” when used in reference to astructural gene refers to the nucleotide sequences which encode theamino acids found in the nascent polypeptide as a result of translationof a mRNA molecule. The coding region is bounded, in eukaryotes, on the5′ side by the nucleotide triplet “ATG” which encodes the initiatormethionine and on the 3′ side by one of the three triplets which specifystop codons (i.e., TAA, TAG, TGA).

As used herein, the term “structural gene” refers to a DNA sequencecoding for RNA or a protein. In contrast, “regulatory genes” arestructural genes that encode products that control the expression ofother genes (e.g., transcription factors).

As used herein, the term “gene” means the deoxyribonucleotide sequencescomprising the coding region of a structural gene and includingsequences located adjacent to the coding region on both the 5′ and 3′ends for a distance of about 1 kb on either end such that the genecorresponds to the length of the full-length mRNA. The sequences whichare located 5′ of the coding region and which are present on the mRNAare referred to as 5′ non-translated sequences. The sequences which arelocated 3′ or downstream of the coding region and which are present onthe mRNA are referred to as 3′ non-translated sequences. The term “gene”encompasses both cDNA and genomic forms of a gene. A genomic form orclone of a gene contains the coding region interrupted with non-codingsequences termed “introns” or “intervening regions” or “interveningsequences.” Introns are segments of a gene which are transcribed intoheterogeneous nuclear RNA (hnRNA); introns may contain regulatoryelements such as enhancers. Introns are removed or “spliced out” fromthe nuclear or primary transcript; introns therefore are absent in themessenger RNA (mRNA) transcript. The mRNA functions during translationto specify the sequence or order of amino acids in a nascentpolypeptide.

In addition to containing introns, genomic forms of a gene may alsoinclude sequences located on both the 5′ and 3′ end of the sequenceswhich are present on the RNA transcript. These sequences are referred toas “flanking” sequences or regions (these flanking sequences are located5′ or 3′ to the non-translated sequences present on the mRNAtranscript). The 5′ flanking region may contain regulatory sequences,such as promoters and enhancers, which control or influence thetranscription of the gene. The 3′ flanking region may contain sequencesthat direct the termination of transcription, posttranscriptionalcleavage and polyadenylation.

The term “label” or “detectable label” are used herein, to refer to anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Such labelsinclude biotin for staining with labeled streptavidin conjugate,magnetic beads (e.g., Dynabeads®), fluorescent dyes (e.g., fluorescein,texas red, rhodamine, green fluorescent protein, and the like),radiolabels (e.g., 3H, 1251, 35S, 14C, or 32P), enzymes (e.g., horseradish peroxidase, alkaline phosphatase and others commonly used in anELISA), and calorimetric labels such as colloidal gold or colored glassor plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.Patents teaching the use of such labels include, but are not limited to,U.S. Pat. No. 3,817,837 [7]; U.S. Pat. No. 3,850,752 [8]; U.S. Pat. No.3,939,350 [9]; U.S. Pat. No. 3,996,345 [10]; U.S. Pat. No. 4,277,437[11]; U.S. Pat. No. 4,275,149 [12]; and U.S. Pat. No. 4,366,241 [13](all herein incorporated by reference). The labels contemplated in thepresent invention may be detected by many methods. For example,radiolabels may be detected using photographic film or scintillationcounters, fluorescent markers may be detected using a photodetector todetect emitted light. Enzymatic labels are typically detected byproviding the enzyme with a substrate and detecting, the reactionproduct produced by the action of the enzyme on the substrate, andcalorimetric labels are detected by simply visualizing the coloredlabel.

The term “small organic molecule” as used herein, refers to any moleculeof a size comparable to those organic molecules generally used inpharmaceuticals. The term excludes biological macromolecules (e.g.,proteins, nucleic acids, etc.). Preferred small organic molecules rangein size from approximately 10 Da up to about 5000 Da, more preferably upto 2000 Da, and most preferably up to about 1000 Da.

DESCRIPTION OF THE FIGURES

The accompanying figures, which are incorporated into and form a part ofthe specification, illustrate several embodiments of the presentinvention and, together with the description, serve to explain theprinciples of the invention. The figures are only for the purpose ofillustrating a preferred embodiment of the invention and are not to beconstrued as limiting the invention.

FIG. 1A shows xrRNA function. Xrn1 (green) loads on a FV RNA, degradesthe RNA, then reaches a discrete ‘xrRNA’ structure in the 3′ UTR andhalts, leading to sfRNAs where Xrn1 appears to remain in an inactivestate.

FIG. 1B shows the pattern of sfRNA production in West Nile Virus(Northern blot).

FIG. 1C shows 3′UTR organization, red arrows are known or putative Xrn1halt sites. Known or putative xrRNAs are boxed. Although depicted asseparate entities, their functions may be coupled. SLs=stem loops,DB=dumbbells.

FIG. 2 depicts the basic architecture of a FV 3′ UTR and proposed haltsites for Xrn1 (DENV2 shown as a representative example). The halt sitesand structures of the ‘SL’ motifs (solid arrows) have beencharacterized; the halt sites and structures of the ‘DB’ motifs remainunexplored (open arrows). Dashed lines indicate likely PK interactions.

FIG. 3 depicts splicing and decay pathway in yeast. Both fully splicedmRNAs and splice defective pre-mRNA intermediates are degraded by Xrn1

FIG. 4A shows the design of reporter transcript with a cricket paralysisvirus (CrPV) IRES, but no xrRNAs. FIG. 4B shows version of thistranscript with xrRNAs (wavy lines) inserted. Transcripts wereconstitutively transcribed from 2-micron plasmids in budding yeast.

FIG. 5A depicts the pathway from transcription to degradation fortranscripts from the non-xrRNA-containing RNAs. FIG. 5B depicts thePathway for the transcripts containing xrRNAs (red). Although bothcontain an IRES (blue) and can produce protein, it was expected that themRNAs without the xrRNA to be degraded more quickly and produce lessprotein, while those with the xrRNAs should be protected fromdegradation and produce more protein.

FIG. 6 shows Protection of translationally competent mRNAs in buddingyeast. Blue denotes the CrpV IRES, red denotes the tandem copies ofxrRNA. The open box is the open reading frame encoding the enzyme. Thelanes are explained in the text. The dashed box indicates the productsthat build up because Xrn1 cannot degrade past the xrRNAs.

FIG. 7 shows an activity assay to measure B-galactosidase (LacZ) levelsmade from mRNAs produced in budding yeast. LacZ actvitiy is reflected byan increase in ONPG hydrolysis (OD420) per cell (OD600). Including thexrRNAs leads to an increase in the amount of LacZ activity,demonstrating that the xrRNA-protected mRNA is competent forIRES-dependent translation. Mutation of the xrRNAs returns LacZ activityto the level of mRNAs without any xrRNAs. This increase in protein isdue to the accumulation of the RNA products boxed in FIG. 6. Theseuncapped, partially degraded, but protected mRNAs are functionaltemplates for translation.

FIG. 8A depicts a secondary structure cartoon of an xrRNA. Thenucteotides explicitly shown are conserved in all xrRNA we have studiedso far. The secondary structural elements are labeled. The P2/L2 stemloop is variable between xrRNAs and we have mutated it without affectingfunction.

FIG. 8B depicts a hypothesized L2 loop as a location where the xrRNAcould be split into two pieces (bottom left sequence and top and rightsequence).

FIG. 8C depicts RNAs used to test this idea. RNA1 (SEQ ID NO: 1) is the“substrate” which is recognized by the 5′ complementary sections of assecond RNA sequence (RNA2), which is the “protector”. In theory, RNA1could be any length, because RNA2 (SEQ ID NO: 2) must only base pair toit as shown. The part labeled as “variable” can be changed to recognizedifferent sequences. The circled nucleotides limit what sequences may berecognized (at this point), but might be varied with more engineering ofthe RNA.

FIG. 8D show the result of a resistance assay. RNA1 is processed to ashorter product, but is not fully degraded by Xrn1 when RNA2 is present.

FIG. 9 shows a cartoon representation of a highly modified engineeredxrRNA-based RNA. The red is the portion that base pairs to any targetmRNA in the cell (dark dashed line) and protects it from degradation. Inlight dashed lines is a plant viral RNA sequence that recruits the capbinding protein (round green ball) and drives translation of theprotected RNA. In dark line on the 3′ end is another RNA structure; itcould be a protein binding site, an aptamer, a Spinach-RNA forvisualizing this RNA in the cell, etc.

FIG. 10 shows a strategy to control Xrn1 resistance using a smallmolecule. In this particular example, the “protecting RNA (solid) has aG-box riboswitch appended to its 3′ end (light dashed line), with asequence that can compete for helix P1 that is formed when theprotecting RNA hybridizes to the substrate RNA (dark dashed line). Uponbinding of the small molecule (block with SM; in this case, hypoxanthineor other related ligands) the riboswitch folds and helix P1′″ forms.This disrupts the P1 helix and the protector” RNA no longer can haltXrn1 from degrading the substrate.

FIG. 11 depicts some types of chemical modification of the RNA that beincluded in the current invention. For example, the chemicalmodification may comprise inclusion of phosphorothioate linkages,boranophosphate linkages, locked nucleic acid, 2′ -modifications, 4′-thio modified RNA, ribo-difluorotoluyl nucleotide, and unchargednucleic acid mimics, as described by Corey (2007) J. Clin. Invest.117(12), 3615-3622 [14], herein encorporated by reference.

FIG. 12 shows that Xrn1 resistant RNAs from flaviviruses can resistdegradation by different exonucleases. FIG. 12 shows a gel where inputRNA containing an xrRNA was challenged with Xrn1, RNase J1 (frombacteria), and Dxo1 (from yeast). All enzymes were expressedrecombinantly and purified to high purity. RppH was included in allreactions to convert the 5′ triphosphate to a monophosphate. Thisdemonstrates that the xrRNA could be engineered to work in diverse hostsand have broad applicability.

DESCRIPTION OF THE INVENTION

Flaviviruses are emerging human pathogens and worldwide health threats.During infection, pathogenic subgenomic flaviviral RNAs (sfRNAs) areproduced by resisting degradation by the 5′ →3′ host cell exonucleaseXrn1 through an RNA structure-based mechanism wherein Xrn1-resistantflaviviral RNA, which contains interwoven pseudoknots within a compactstructure that depends on highly conserved nucleotides [15]. The RNA' sthree-dimensional topology creates a ring like conformation, with the 5′end of the resistant structure passing through the ring from one side ofthe fold to the other. Disruption of this structure prevents formationof sfRNA during flaviviral infection. Thus, sfRNA formation results froman RNA fold that interacts directly with Xrn1, presenting the enzymewith a structure that confounds its helicase activity. The currentinvention envisions adapting creating similar RNA based interwovenpseudoknot structures to enable the preservation of and expression oftargeted RNA transcripts.

Signal Activated RNA Interference

One reference, U.S. Pat. No. 9,029,524 [16], describes compositions andmethods for signal activated RNA interference (saRNAi). The referencestates that a pseudoknot is employed and may be resolved by theintroduction of signal, such as a second polynucleotide. Unknotting ofthe pseudoknot may be accomplished when a signal polynucleotide binds toone of three regions on the signal-activated polynucleotide. In oneexample, the signal polynucleotide binds to the signal-detectingnucleotide and displaces the pseudoknotting strand from the stem-loopstructure. This exposes the stem-loop structure to cleavage by Dicer andprocessing by the RISC complex. In a second example, the signalpolynucleotide binds to the signal-detecting nucleotide at a regionupstream of the pseudoknot, causing compression and steric/electrostaticrepulsion between the signal-detecting strand and the stem-loopstructure. The reference describes the polynucleotide structures assubstantially resistant to endonucleases and exonucleases. The referencedoes not describe xrRNA sequences that contain a second heterologoussequence, nor does it describe the hybridization of two separate RNAmolecules to form such an interwoven pseudoknot structure.

One reference, Chapman, E. G. et al. (2014), Science 344(6181), 307-310[15], describes the structure of the sfRNA which halts the advance ofthe Xrn1 endonuclease via interruption of the Xrn helicase. Thereference describes the crystallized structure of the sfRNA ascontaining interwoven pseudoknots that interrupt the structure throughsequence changes, and halts the effectiveness of Xrm1. The RNA sequencesdescribed are sections of naturally occurring xrRNA and those that wereengineered to evaluate the sequence's effectiveness against Xrn1. Thereference concludes that the xrRNA structure is braced against theenzyme, holding the leading edge of the relevant RNA duplex behind theconcave ring structure and away from these helices, suggesting amechanism for RNA structure-driven Xrn1 resistance and, by extension,sfRNA production. The reference does not describe xrRNA sequences thatcontain a second heterologous sequence, nor does it describehybridization of two separate RNA molecules to form such an interwovenpseudoknot structure.

One reference, Chapman, E. G. et al. (2014), eLife 3, e01892 [17],describes modification of sfRNA to evaluate the sequence and structuralfeatures of the RNA that conferred resistance to the Xrn1 endonuclease.The reference concludes that disruption of the lead sequences of sfRNAleads to the elimination of disease related sfRNAs, thus linking the RNAstructure to sfRNA production. The reference states that suchXrn1-resistant activity resides in discrete and portable structuralelements, the RNA sequences. The reference does not describe xrRNAsequences that contain a second heterologous sequence, nor does itdescribe the hybridization of two separate RNA molecules to form such aninterwoven pseudoknot structure.

One reference, Kieft, J. S. et al. (2015), RNA Biol. E-published Sep.23, 2015 [18], describes that continued analysis and interpretation ofthe structure of sfRNAs reveals that the tertiary contacts that knit thexrRNA fold together are shared by a wide variety of arthropod-borne FVs,conferring robust Xrn1 resistance in all tested. The reference furtherdiscloses that some variability in the structures that correlates withunexplained patterns in the viral 3′ UTRs. The references' examinationof these structures and their behavior in the context of viral infectionleads to a new hypothesis linking RNA tertiary structure, overall 3′ UTRarchitecture, sfRNA production, and host adaptation. The reference doesnot describe xrRNA sequences that contain a second heterologoussequence, nor does it describe the hybridization of two separate RNAmolecules to form such an interwoven pseudoknot structure.

One reference, Moon, S. L. et al. (2012), RNA 18(11), 2029-2040 [19],describes an evaluation of sfRNAs, including several experimentsintroducing sfRNA sequences downstream from a reporter GFP-expressingconstruct (e.g., a heterologous sequence). In part, this was done toevaluate the decay curves for mRNA in correlation with the amount ofsfRNA present in a cell. These experiments further establish therelationship of sfRNA with XRN1 depletion and mRNA transcript stability.The reference states that pseudoknots and related structures have beenshown in the past to hinder the action of other enzymatic complexes thatmove directionally along RNA and that pseudoknots can serve asroadblocks for ribosomes and influence frameshifting rates. Thereference does not describe sfRNA sequences upstream of heterologousRNA. The reference does not describe xrRNA sequences that contain asecond heterologous sequence, nor does it describe the hybridization oftwo separate RNA molecules to form such an interwoven pseudoknotstructure.

One reference, Burke, D. H. et al. (1996), J. Mol. Biol. 264(4), 650-666[20], describes several RNA inhibitors of HIV-1 RT that differsignificantly from the pseudoknot ligands found previously, along with awide variety of pseudoknot variants. Patterns of conserved and covaryingnucleotides yielded structural models consistent with 5′ and 3′ boundarydeterminations for these molecules. Among the four isolates studied indetail, the first is confirmed as being a pseudoknot, albeit withsubstantial structural differences as compared to the canonicalpseudoknots identified previously. The second forms a stem-loopstructure with additional flanking sequences required for binding. Theminimal fully active truncations of each of these four isolates competewith each other and with a classical RNA pseudoknot for binding to HIVRT, suggesting that they all recognize the same or overlapping sites onthe protein, in spite of their apparently dissimilar structures. Thereference does not describe xrRNA sequences that contain a secondheterologous sequence, nor does it describe the hybridization of twoseparate RNA molecules to form such an interwoven pseudoknot structure.

One reference, U.S. Pat. No. 5,256,775 [21], describes making 3′ and/or5′ end-capped oligonucleotides so as to render the oligonucleotideresistant to degradation by exonucleases. The exonuclease degradationresistance is provided by incorporating two or more phosphoramidate andphosphorocmonothioate and/or phosphorodithioate linkages at the 5′and/or 3′ ends of the oligonucleotide, wherein the number ofphosphoramidate linkages is less than a number which would interferewith hybridization to a complementary oligonucleotide strand and/orwhich would interfere with RNAseH activity when the oligonucleotide ishybridized to RNA. The reference does not describe xrRNA sequences orinterwoven pseudoknots, nor does it describe the hybridization of twoseparate RNA molecules to form such an interwoven pseudoknot structure.

One reference, U.S. Patent Application Publication Number US2014-0329880 A1 [22], describes exonuclease resistant polynucleotideswith a 5′ end and a 3′ end and comprises a blocker domain having anon-nucleic acid polymer segment and a phosphorothioate segment. Thereference also describes exonuclease resistant duplex polynucleotidehaving a length of about 17 to about 30 bp and comprising a guide strandcomplementary bound to a passenger strand, each of the guide strand andpassenger strand having a 5′ end and a 3′ end, the duplex RNA having atleast one configuration allowing processing of the guide strand by dicerand/or an argonaute enzyme, the passenger strand comprising theexonuclease resistant polynucleotide herein described, in aconfiguration in which the second end of non-nucleic acid polymer ispresented at the 5′ end of the passenger strand. In some embodiments,the exonuclease resistant duplex polynucleotide is a targeting domain.The reference does not describe xrRNA sequences or interwovenpseudoknots, nor does it describe the hybridization of two separate RNAmolecules to form such an interwoven pseudoknot structure.

Various Embodiments

In one embodiment, the invention contemplates various applications ofRNA sequences derived from the genomic RNA of flaviviruses (FVs). Thesederived RNA sequences have been found to be resistant to cell enzymeXrn1, the dominant cytoplasmic 5′→3′ exonuclease. These discreteXrn1-halting sequences are referred to as “Xrn1-resistant RNAs”(xrRNAs). The xrRNAs have been found to have distinct structuralelements and further have certain conserved sequences to retain theseresistant structural elements.

In one embodiment, the invention contemplates that such sequences of RNAcould be useful in a number of applications including, but not limitedto: A) introduction of an xrRNA sequence “in cis” upstream of desiredheterologous mRNA sequences to improve the half-life of the heterologousmRNA sequences from exonuclease degradation; and B) provide Xrn1nuclease resistance by the “in trans” association of an RNA thatprovides Xrn1-halting ability to an RNA that does not have this ability,thus protecting a “target” RNA from degradation by hybridization of a“protecting” RNA. In this approach, structures similar to xrRNAs couldbe generated by the hybrid association of two heterologous RNAs ratherthan placing an xrRNA sequence on at least one heterologous RNAsequence.

In one embodiment, the invention contemplates that the “in trans”configuration could be used: (1) to alter specific endogenous RNAdegradation rates within cells as a “transcriptome editing” tool and (2)coupling such RNA combinations with a way to turn protected (butuncapped or decapped) mRNAs into viable translation templates as a“translatome editing” tool. It is believed by adjustment of the RNAsequence, that the “protecting RNA” could be “tuned” to achieve variousprotection levels and translation levels of endogenous RNAs.

DETAILED DESCRIPTION OF THE INVENTION

The technology/inventions described here are based on our discoveriesregarding a viral RNA that has the extraordinary ability to block apowerful cellular exonuclease. This RNA structure is thought to becritical to the virus, but the current invention has now enabled theremoval of the RNA structure from that context and engineer it as amanipulator of cellular processes.

Initial studies were focused on the ability of flaviviruses (FVs) to useRNA structure to resist degradation by Xrn1, a powerful cellularexonuclease. Briefly, during replication of arthropod-borne FVs, copiesof the viral genomic RNA are made. Infection also leads to a set ofdiscrete shorter flaviviral RNAs that accumulate to high levels [23-25].These subgenomic flaviviral RNAs (sfRNAs) are −200-500 nt in length andare directly linked to viral cytopathicity in cultured cells (mammalianand insect) and for disease symptoms in fetal mice [26-28]. The sfRNAsare made by partial digestion of FV genomic RNA by host cell enzymeXrn1, the dominant cytoplasmic 5′→3′ exonuclease (FIG. 1) [27]. Innormal cellular metabolism, Xrn1 recognizes the 5′ monophosphate ofdecapped mRNAs and catalyzes their degradation in a 5′ to 3′ directionalprocess [29]. In FV infection, Xrn1 loads on some presumably decappedcopies of the viral RNA genome and degrades them in a 5′→3′ processuntil it halts at defined locations in the 3′UTR, resulting in the setof sfRNAs [26-28]. This mechanism is surprising given Xrn1's ability todegrade stable structured RNAs such as ribosomal RNA. These discreteXrn1-halting elements are referred to as “Xrn1resistant RNAs”(xrRNAs)[17]. An FV xrRNA has been characterized, that being thestem-loop (SL) type often found in tandem near the 5′ end of FV 3′UTRs[26-28] (FIG. 2). The functional, biochemical, structural, andvirological studies included the first high-resolution three-dimensionalstructure of a functional SL-type xrRNA (solved by crystallography). Itwas found that these RNAs adopt a specific three-dimensional fold, andthere is a suggested mechanism for how these RNAs stop progression ofXrn1 using a combination of thermodynamic stability coupled to a uniquefold that mechanically confounds the helicase activity of Xrn1. Thischaracterization of these RNAs has revealed how novel and remarkablethey are; the structure is unlike anything previously found. Thesediscoveries were published in 2014 in Science and eLife, the contents ofwhich are incorporated herein by reference [15, 17].

These discoveries have provided an insight into the function of thesexrRNAs and also inspired the current invention ideas for how they couldbe used as tools, potentially as part of novel therapeutic strategies inwhich stabilizing RNAs within a cell is important. In other words, noway is known wherein a specific RNA within a cell can be super-stable,perhaps evading the degradation machinery completely and remaining aviable, biologically active RNA. The xrRNAs may provide the basis forcreative engineering and inventions that would allow this type ofprecise control within living cells. The current invention engineers andexplores this possibility.

Demonstration that Flavivirus-Derived xrRNAs Can Resist Xrn1 in LivingCells to Protect Functional mRNAs

Characterization of these xrRNAs demonstrated that they appear to bemodular structure elements. That is, the current invention hypothesizesthat one could remove an xrRNA sequence from its native context in theviral genomic RNA and place it within any other RNA, and thus protectany downstream sequences from degradation by Xrn1. If true, one couldthen extend the lifetime of any RNA in the cell in which this elementwas installed. To test this, the Xrn1-mediated decay process in yeastwas chosen as a tractable model system (FIG. 3). A vector that woulddrive expression of an mRNA was created containing (from 5′ to 3′):leaders sequence, the ACT1 intron, a viral internal ribosome entry site(IRES), and a LacZ reporter sequence (FIG. 4A). The viral IRES wasincluded because partial digestion of the mRNA by Xrn1 may give rise toa stable, but uncapped message that would not be used in translation;the IRES allows translation to occur internally, downstream of thexrRNAs. Thus, in this mRNA two viral RNA structures from differentviruses have been coupled. Expression of this mRNA in yeast may giverise to a spliced and capped RNA that would serve as the template forproducing the reporter proteins at some level, and would also be subjectto degradation at some rate (FIG. 5A & B). To test the function of thexrRNAs, the two “tandem” xrRNAs from Dengue virus were installed intothis reporter construct between the intron and the IRES (FIG. 4B). Ifthe xrRNAs protect the message from Xrn1 degradation, one might expectan accumulation of partially degraded mRNAs that should be able toproduce enzyme, driven by the action of the IRES (FIG. 5A & B).

This experiment was successful, and is shown in FIG. 6 and FIG. 7.

This idea was also used to test the Dbr1/Xrn1-mediated decay ofsplice-defective pre-mRNA intermediates and found results that areconsistent with the above.

The important conclusions: 1) The xrRNAs from flaviviruses are modularelements that may be placed in a non-native context where they retainfunction. 2) In living cells that differ from those they evolved in,these xrRNAs halt Xrn1 and protect downstream RNA. 3) The protected RNAsare competent as templates for translation, in this case using an IRESplaced downstream of the xrRNAs.

These results suggest that xrRNAs may be used as powerful genetic toolsto artificially ex1end the lifetime of any arbitrary RNA within a livingcell. If placed within an mRNA, they may protect anything that is 3′ ofthem, and other RNA elements can be engineered with the xrRNAs toachieve specific outcome. This could be achieved using modern genomicediting tools such as CRISPR to install these engineered elements in anyRNA in a cell. It is believed that currently, there is no existing wayto extend the lifetime of a specific arbitrary RNA in cells. Althoughthis method has only been tested in yeast, the fact that xrRNAs work inboth insect and mammalian cells during flavivirus infection suggeststhat they may in fact be “universal” Xrn1 blockers. Furthermore, theIRES used in these studies has been shown to function in diversesystems, including human cells, yeast, rabbit reticulocyte lysate,bacteria, wheat germ extract, insects, etc. Hence, this may be a widelyapplicable method.

Demonstration that Xrn1 Resistance Can be Achieved by in TransAssociation of Two RNAs.

The results described above show that the xrRNAs could be a powerfulgenetic tool. In the manifestation described in section II, the xRNA wasplaced within the RNA to be protected (i.e. placed in cis). This isuseful, but the power of the xrRNAs could be extended if they couldsomehow be used in a method in which one RNA could protect another RNAin trans. Towards this end, the published crystal structure of an xrRNAwas examined and published biochemical characterization results alsoconsidered [15, 17]. These data show that loop L2 of the xrRNA structuremay be mutated without functional affect, and the P2 stem that this loopcaps may be shortened, lengthened, or altered without losing the abilityto halt Xrn1 (FIG. 8A & B). It was therefore reasoned that one couldsplit the xrRNA into two pieces: a “target” strand (RNA1) and a“protecting” strand (RNA2). It was hypothesized that in the absence ofRNA2, RNA would be rapidly degraded by Xrn1, but when RNA2 was added, itwould anneal to RNA1, induce a fold, and protect RNA1 from degradation(FIG. 8C). This idea was tested using the established in vitro systemwith purified Xrn1 (FIG. 8D). As hypothesized, RNA1 (SEQ ID NO: 1,GGGCCGGCAAAACUAACAUGAAAACAAGGCUAAAAGCCAGGUCGGAUUACCCUUUUGGAUCCCGACUGGCGAGAGCCA) was able to protect by addition of RNA2 (SEQ IDNO: 2, GGGUAAUCCGCCAUAGUACGGAAAAAACUAUGCUACCUGUGAGCCCCGUCCAAG GACGUU),demonstrating the current invention idea of engineering the xrRNA tooperate as an in trans protector is a valid approach. This result showsthat the current invention may be an RNA-based on an xrRNA, which offersin trans protection to other RNAs from degradation by Xrn1. Such an RNAhas not been discovered in nature.

The potential uses and implications of this invention are many.Currently, it has been shown that this system works in vitro. If the RNAcan be shown to work in the complex environment of living cells, onecould have a way to protect endogenous, unmodified RNAs from degradationwithin a cell. This is exciting, because mRNA-based therapeutics arebeing actively pursued by several entities, but one limitation/challengethey face is the stability of the therapeutic mRNAs. In addition, onemight be able to increase the levels of desirable mRNAs such asantitumor-encoding mRNAs or even noncoding RNAs with desirable effects,without having to alter the genome. As CRISPR is a genome-editing tool,the technology of the current invention could be a transcriptome-editingtool.

Development of “In Trans” Protection of RNA Method

The creation of an “in trans” protection system in vitro describedherein is exciting, but additional value lies in:

(1) Using the current invention system in more complex environments toalter specific endogenous RNA degradation rates within cells as a“transcriptome editing” tool.

(2) Coupling it with a way to turn protected (but uncapped) mRNAs intoviable translation templates as a “translatome-editing” tool.

Steps for achieving this and for simultaneously developing additional invitro and in vivo tools are described below.

Step 1: Demonstrate that in trans protection can yield a stable messagethat is a substrate for translation in lysate, using an IRES installedwithin the mRNA. This is similar to what was done in yeast, except thexrRNA will not be installed in the message; rather protection will beprovided in trans.

Step 2: Develop a way to provide the signal to initiate translation intrans, coupled to the protecting RNA An endogenous cellular mRNA has noIRES, and thus partial degradation by Xrn1 would not yield a viabletemplate for translation. However, some plant viruses use a discretestructure in their 3′ untranslated regions that binds the cap-bindingprotein e1F4E without using a cap. This structure is brought to the 5′end of the plant viral RNA by base-pairing, and this leads totranslation initiation (FIG. 9) [30]. Hence, nature has provided atranslation initiation element that we may exploit to be used in trans.It is thought that by coupling this to the current xrRNA invention, onemay produce a single RNA that can protect another RNA in trans AND driveits translation.

Step 3. Test functionality in living cells. The above ideas (steps 1 and2) can first be tested in a cell-free, translationally competent lysateusing added Xrn1. Once success is achieved in these systems, thefunctionality of these RNAs will be tested by transfecting them orexpressing them in human cell culture, targeting various endogenousmRNAs. If one could add an RNA to a cell, extend the lifetime of aspecific RNA this would be very powerful (the opposite of siRNAs orshRNAs), this would be a novel method of broad usefulness. If the RNA isan mRNA, and one can drive it to be translated, this would also be verypowerful. Achieving this would be a major leap, with consequences forboth research tools and therapeutics.

Step 4. Extend the RNA targets that can be affected by xrRNAs.Currently, the potential use of the in trans protection method islimited by the fact that the “protecting RNA” only can pair to a certainsequence in the target RNA; thus, this sequence must be naturally foundin the endogenous RNA This sequence is not overly constraining (forexample, the P53 5′ leader contains it) but does limit use. Thus,efforts could be made to engineer the protecting RNA through severalstrategies (including characterizing additional viral xrRNAs and invitro selection methods) to expand the list of targets.

Step 5. “Tune” the “protecting RNA” to achieve various protection levelsand translation levels of endogenous RNAs.

Although each of these steps are in progress, they represent anintellectual leap in taking an RNA found in the 3′ end of flavivirusesand using it to develop a tool that can be used to protect an arbitraryRNA in a cell from degradation by Xrn1, driving its translation (ifdesired), and doing so in a tunable way with no need to overexpress thetarget RNA or edit the genome.

xrRNAs Could Be Controlled By Small Molecules

An additional level of functionality to the above-described method couldbe achieved if the protecting function was controllable by the action ofa small molecule. This could be achieved in both the “in cis” or “intrans” xrRNAs. To develop this, two next steps are envisioned:

Step 1. Demonstrate the ability to couple an xrRNA to a riboswitch.Riboswitches are metabolite-sensing RNAs that change their folddepending on the binding off the small molecule metabolite. They couldbe used to create a small molecule sensing xrRNA, one way that is likelyto work (a purine-sensing riboswitch [31]) is shown in FIG. 10. The useof riboswitches might allow coupling of a given mRNA's abundance tometabolite concentrations.

Step 2. The use of riboswitches to control xrRNA function is useful, butfor many applications is limited because ultimately there is a desire tocontrol the xrRNA with a small molecule that is NOT an endogenousmetabolite. Selection-based, directed evolution-type methods can helpachieve this. The example shown in FIG. 9 is a proof of principle;long-term engineering could yield version capable of responding to a setof small molecules either positively or negatively.

xrRNAs could be used to simultaneously protect RNA and deliver “cargo”

The fact that the P2/L2 stem-loop in an xrRNA can be altered extensivelywithout functional consequence suggests that this could be a place toinstall specific RNA sequences as “cargo” that would be delivered withthe xrRNA. Likewise, the 5′ or 3′ ends of the RNA could be used toattached additional cargo (as with the e1F4E binding element mentionedabove). For example, a protein-binding sequence could be placed there,or “Spinach” sequence [32, 33], or even perhaps a short open readingframe (FIG. 9). Cargo could also be attached to the 3′ end of theprotecting RNA, which is not necessary for protection from Xrn1. In thisway, an RNA could be protected and coupled with delivery of a specificRNA structure or functional element. In its most dramatic manifestation,an entire open reading frame could be placed as cargo, and combined withsmall-molecule dependent control (for example) unique tools created.

xrRNAs Could Be Used to Block Diverse Exonucleases and Affect RNA LevelsIn Several Domains of Life

Flaviviruses infect eukaryotes and the xrRNAs have evolved to resistprogression of Xrn1. However, it was reasoned that they might functionby forming a general mechanical unfolding blocks that would stopexonucleases in addition to Xrn1. The ability of an xrRNA to block RNaseJ1, a 5′→3′ exonuclease from bacteria with no known structural homologyto Xrn1, was tested. The xrRNA was able to block bacterial RNase J1 andother enzymes (see FIG. 12) suggesting that the technology developed anddescribed herein could be broadly applied.

Chemical Modification of the RNA

There are many methods of chemical modification of RNA known in the art.In some embodiments, the chemical modification may comprise chemicalmodification of one xrRNA containing RNA sequence. In one embodiment,wherein there is a substrate strand and a protecting strand, one or bothstrands may be chemically modified. Although not limiting the currentinvention to any particular means or type of chemical modification, inone embodiment, chemical modification comprises modification of the 3′or 5′ ends of said RNA strands. In one embodiment, the 5′ modificationmay include: addition of 7-methylguanosine (m7G) and other syntheticadditions. In one embodiment, the 3′ modification may include: additionof a poly(A) tail and other synthetic additions

In some embodiments, chemical modification of RNA may also compriseinclusion of phosphorothioate linkages, boranophosphate linkages, lockednucleic acid, 2′-modifications, 4′-thio modified RNA,ribo-difluorotoluyl nucleotide, and uncharged nucleic acid mimics, asdescribed by Corey 2007 J. Clin. Invest. 117(12), 3615-3622 [14], hereinencorporated by reference. See FIG. 11. Replacing one nonbridging oxygenatom on the backbone phosphate between two ribonucleotides with asulphur atom creates a phosphorothioate (PS) linkage. In one embodiment,the current invention contemplates modifying the phosphate backbone ofan oligonucleotide is the introduction of a boron atom in place of oneof the nonbridging oxygen atoms to create a boron-phosphorous linkage.In one embodiment, the current invention contemplates the use of lockednucleic acid (LNA) nucleotides that contain a methylene bridge betweenthe 2′ and 4′ carbons of the ribose ring. In one embodiment, the currentinvention contemplates the use of substitutions for the hydroxyl groupon the 2′ carbon atom of the ribose ring of particular nucleotides. Inone embodiment, the current invention contemplates the use of 4′-Thiomodified nucleotides, which contain a sulphur atom in place of oxygenattached to the 4′ carbon of the ribose ring. In one embodiment, thecurrent invention contemplates introduction of ribo-difluorotoluyl (rF)nucleotides (65). In one embodiment, single-stranded uncharged nucleicacid mimics, such as peptide nucleic acids (PNAs) (66, 67) andmorpholino oligomers (68) may be useful as partial or full replacement sfor nucleotides in the RNA.

Thus, specific compositions and methods of protecting RNAs fromdegradation using engineered viral RNAs have been disclosed. It shouldbe apparent, however, to those skilled in the art that many moremodifications besides those already described are possible withoutdeparting from the inventive concepts herein. Moreover, in interpretingthe disclosure, all terms should be interpreted in the broadest possiblemanner consistent with the context. In particular, the terms “comprises”and “comprising” should be interpreted as referring to elements,components, or steps in a non-exclusive manner, indicating that thereferenced elements, components, or steps may be present, or utilized,or combined with other elements, components, or steps that are notexpressly referenced.

Although the invention has been described with reference to thesepreferred embodiments, other embodiments can achieve the same results.Variations and modifications of the present invention will be obvious tothose skilled in the art and it is intended to cover in the appendedclaims all such modifications and equivalents. The entire disclosures ofall applications, patents, and publications cited above, and of thecorresponding application are hereby incorporated by reference.

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We claim:
 1. A synthetic ribonucleic acid (RNA) sequence comprising anexonuclease resistant RNA sequence ligated to a 5′ end of a heterologousRNA sequence, wherein said exonuclease resistant RNA sequence comprisesan interwoven pseudoknot structure.
 2. The synthetic RNA sequence ofclaim 1, wherein said interwoven pseudoknot structure comprises aconserved three-way junction.
 3. The synthetic RNA sequence of claim 1,wherein said synthetic RNA sequence further comprises an internalribosome entry site sequence between said exonuclease resistant RNAsequence and said heterologous RNA sequence.
 4. The synthetic RNAsequence of claim 1, wherein said synthetic RNA sequence furthercomprises at least one chemical modification at either the 5′-end or3′-end.
 5. The synthetic RNA sequence of claim 1, wherein said syntheticRNA sequence further comprises at least one chemically modifiednucleotide.
 6. A synthetic ribonucleic acid (RNA) duplex comprising afirst exonuclease resistant RNA sequence hybridized to a secondheterologous RNA sequence, wherein a region of said first exonucleaseresistant RNA sequence and a region of said second heterologous RNAsequence comprise an interwoven pseudoknot structure.
 7. The syntheticRNA duplex of claim 6, wherein said interwoven pseudoknot structurecomprises a conserved three-way junction.
 8. The synthetic RNA duplex ofclaim 6, wherein said second heterologous RNA sequence comprises anaturally occurring RNA sequence.
 9. The synthetic RNA duplex of claim6, wherein said first exonuclease resistant RNA sequence furthercomprises a heterologous RNA sequence ligated to the 3′ end of saidexonuclease resistant RNA sequence.
 10. The synthetic RNA duplex ofclaim 9, wherein said heterologous RNA sequence comprises small moleculesensing riboswitch.
 11. The synthetic RNA duplex of claim 9, whereinsaid first sequence further comprises a translation initiation element.12. The synthetic RNA duplex of claim 10, wherein said riboswitchdisrupts the interwoven pseudoknot structure in the presence of saidsmall molecule.
 13. The synthetic RNA duplex of claim 9, wherein saidheterologous RNA sequence comprises an open reading frame.
 14. Thesynthetic RNA duplex of claim 9, wherein said heterologous RNA sequencecomprises a protein binding sequence.
 15. The synthetic RNA duplex ofclaim 9, wherein said heterologous RNA sequence comprises a spinachsequence.
 16. The synthetic RNA duplex of claim 6, wherein saidsynthetic RNA duplex comprises a chemical modification of either the 5′end or 3′ end.
 17. The synthetic RNA duplex of claim 6, wherein saidsynthetic RNA duplex comprises at least one chemically modifiednucleotide.